Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: Effect of N-terminus in thermal activity and stability

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the Delta N16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 degrees C for Delta N16 and Delta N26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 degrees C, and maximal stability at pH 8.1 and 65 degrees C. KLEST-5A (Delta N26) did not show an absolute maximum of activity. However, best results were obtained at 40 degrees C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 degrees C (t(1/2) < 5 min) than the wild-type enzyme (t(1/2) = 135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(1/2) = 230 min, at 85 degrees C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability. (C) 2011 Elsevier Inc. All rights reserved.